This invention relates to disc assay devices, systems, and methods of use for culturing biological samples to detect and/or enumerate microorganisms present within the samples.
The detection and enumeration of microorganisms is practiced in numerous settings, including the food-processing industry (testing for the contamination of food by microorganisms such as E. coli and S. aureus), the health care industry (testing of patient samples and other clinical samples for infection or contamination), environmental testing industry, the pharmaceutical industry, and the cosmetic industry.
Growth-based detection and enumeration of microorganisms is commonly practiced using either liquid nutrient media (most probable number analysis (MPN)) or semi-solid nutrient media (agar petri dishes). Enumeration using the liquid MPN method is typically achieved by placing serial 10-fold dilutions of a sample of interest in replicate sets of tubes containing selective media and chemical indicators. The tubes are incubated at elevated temperature (24-48 hours) followed by examination for growth of organisms. A statistical formula, based on the volume of sample tested and the number of positive and negative tubes for each set, is used to estimate the number of organisms present in the initial sample.
This method of performing MPN analysis has several disadvantages. It is labor intensive because of the multiple diluting and pipetting steps necessary to perform the analysis. In addition, in practice it is only practical to use replicate sets of about three to five tubes for each dilution. As a result, the 95% confidence limits for an MPN estimate for microbial concentration are extremely wide. For example, a three tube MPN estimate of 20 has 95% confidence limits ranging from 7 to 89.
In contrast to the method described above, a direct count of viable microorganisms in a sample can be achieved by spreading the sample over a defined area using nutrient media containing a gelling agent. The gelling agent (agar) prevents diffusion of the organisms during incubation (24-48 hours), producing a colony in the area where the original organism was deposited. There is, however, a limit to the number of colonies that can fit on a given area of nutrient media before fusion with neighboring colonies makes counting difficult. This usually necessitates performing several dilutions for each sample. In addition, the classes of chemical indicator molecules that can be used for identifying individual types of microorganisms present within a mixed population are limited to those that produce a product that is insoluble in the gelled media.
In some of these processes, the detection or enumeration of a microorganism is determined by detecting an electromagnetic signal, e.g., fluorescence, emitted by an indicator substance in response to excitation (where the indicator substance is activated by presence of the microorganism to be detected). The excitation may be provided in the form of electromagnetic energy from, e.g., a laser. One potential problem with known assay devices is that the electromagnetic energy used for excitation may also excite other materials present in the substrate or other portions of the device, causing them to emit an electromagnetic signal similar to that emitted by the desired indicator. For example, where the assay is formed on a polymeric substrate that fluoresces in the same or similar wavelength regions as the indicator, a relatively high background electromagnetic signal can be produced by the substrate that reduces the signal-to-background ratio. A lower signal-to-background ratio can make accurate detection or enumeration of the desired microorganism more difficult.
The invention provides devices and methods for the detection and enumeration of microorganisms. The devices and methods include a plurality of discs attached to a substrate and a reflector proximate one surface of each of the discs to reflect electromagnetic energy of selected wavelengths after the energy has passed through the disc. The reflector may be useful to improve the accuracy of detection and/or enumeration of target microorganisms on the assay devices. A system for detecting and/or enumerating target microorganisms on disc assay devices is also provided.
One advantage of the devices and methods of the present invention is that the reflectors proximate each of the discs may reflect the excitation energy directed at the discs after it has passed through the discs. As a result, the effective path length of the excitation energy through the discs is doubled as compared to assay devices in which the excitation energy is not reflected after passing through the discs. That increased path length may improve the accuracy of the detection and/or enumeration of microorganisms by effectively increasing the intensity of the excitation energy.
Another potential advantage of the devices and methods of the invention is that the reflectors may improve the signal-to-background ratio during detection and/or enumeration of target microorganisms. In some instances, the reflectors preferably reduce or prevent transmission of the excitation energy to the underlying substrate. In other instances, the reflectors may reduce or prevent the transmission of electromagnetic energy from, e.g., the substrate, that is similar to the electromagnetic signal energy emitted by, e.g., an indicator substance, in response to the excitation energy. In either case, with the reflectors in place, the electromagnetic signal energy emitted from the assay device may more accurately indicate the presence of the target microorganisms, rather than the underlying substrate or other portions of the device.
The signal-to-background ratio may be further improved if the reflectors are also reflective for the electromagnetic signal energy generated as a result of the presence of the target microorganisms within or on the discs. If the reflectors are located on the side of the discs opposite the detector, at least a portion of the electromagnetic signal energy that is emitted in the direction of the reflectors may be reflected back towards the detector, thereby potentially increasing the intensity of the emitted signal energy. Increases in emitted signal energy intensity may improve detection accuracy and, in some instances, may also allow for detection of the target microorganisms after shorter incubation periods.
With respect to the excitation/detection systems of the present invention which include reflective walls, one potential advantage is that the reflectivity of the walls may improve uniformity in excitation of the assay devices being sampled.
In one aspect, the present invention provides a microorganism culture device including a substrate; a plurality of liquid-retaining discs attached to the substrate, wherein each of the plurality of discs has first and second opposing surfaces; and a reflector proximate the first surface of each of the plurality of discs, wherein electromagnetic energy of selected wavelengths is reflected from the reflector after passing through the disc.
In another aspect, the present invention provides a method of detecting at least one target microorganism by providing a disc assay device including a substrate; a plurality of liquid-retaining discs attached to the substrate, each of the plurality of discs having first and second opposing surfaces; and a reflector proximate the first surface of each of the plurality of discs; and wherein at least one of the discs further includes growth media, an indicator substance, and a target microorganism; directing excitation energy at the disc assay device; and detecting signal energy emitted from each of the plurality of discs including the target microorganism in response to the excitation energy; wherein at least a portion of the signal energy is reflected by the reflector.
In another aspect, the present invention provides a method of manufacturing a disc assay device by providing a substrate; locating a plurality of liquid-retaining discs on the substrate, each of the plurality of discs having first and second opposing surfaces; and providing a reflector proximate the first surface of each of the plurality of discs, wherein electromagnetic energy of selected wavelengths is reflected from the reflector after passing through the disc.
In another aspect, the present invention provides a system for detecting target microorganisms on a disc assay device, the system including at least one excitation source emitting electromagnetic excitation energy of selected wavelengths; a detection chamber including at least one wall, wherein the at least one wall reflects a significant portion of the electromagnetic excitation energy incident thereon; and a detector arranged within the detection chamber to detect electromagnetic signal energy emitted from the assay device in response to the electromagnetic excitation energy.
These and other features and advantages of the invention will be described in connection with illustrative embodiments of the invention below.
As used herein, the term xe2x80x9cmicroorganismxe2x80x9d includes all microscopic living organisms and cells, including without limitation bacteria, mycoplasmas, rickettsias, spirochetes, yeasts, molds, protozoans, as well as microscopic forms of eukaryotic cells, for example single cells (cultured or derived directly from a tissue or organ) or small clumps of cells. Microorganisms are detected and/or enumerated not only when whole cells are detected directly, but also when such cells are detected indirectly, such as through detection or quantitation of cell fragments, cell-derived biological molecules, or cell by-products.